Method for inhibiting replication of HIV

ABSTRACT

A method for inhibiting replication of HIV, which comprises subjecting HIV-infected cells to a composition (a) comprising as a primary component a polysaccharide derived from a hot aqueous solvent extract of tubercle bacillus.

BACKGROUND OF THE INVENTION

1. Field of the Invention:

The present invention relates to a method for the inhibition of HIV(human immunodeficiency virus) replication.

2. Discussion of the Background

AIDS is a disease caused by an HIV infection, and the number of patientssuffering from this disease has markedly increased in recent years. Thetherapy for AIDS has involved use of nucleoside-type anti-HIV agents,such as Zidovudine (Azidothymidine, AZT) and Didanosine (ddI).

Unfortunately, these conventional anti-HIV agents do not provide asufficient therapeutic effect. Consequently, development of a newanti-HIV agent which exhibits an enhanced, and thus adequate,therapeutic effect is required.

More recently, the present inventors discovered and have described inU.S. Ser. No. 09/038,041, that the therapeutic effect of nucleoside-typeanti-HIV agents on AIDS patients may be improved by the combined use ofa hot water extract of human tubercle bacillus and the agents. Thiscomposition was found to reduce the mortality of mice with AIDS ascompared to the anti-HIV agent being administered alone.

However, a need for a simple and effective therapeutic agent forinhibiting the replication of HIV continues to exist.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide asimple and effective agent affording a sufficient therapeutic effect forinhibiting replication of HIV. This method will entail subjectingHIV-infected cells to a composition containing as a primary component apolysaccharide derived from a hot aqueous solvent extract of tuberclebacillus.

It is also an object of the present invention to provide an anti-HIVagent, which contains the above-mentioned composition as an activeingredient.

Yet another object of the present invention is to provide an anti-HIVcomposition containing the above-mentioned composition.

The above objects and others are provided by a method for inhibitingreplication of HIV, which entails subjecting HIV-infected cells to acomposition containing as a primary component a polysaccharide obtainedfrom a hot aqueous solvent extract of tubercle bacillus.

BRIEF DESCRIPTION OF DRAWINGS

The drawing illustrates the anti-HIV effect of the composition of thepresent invention on the replication of HIV in cultures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated upon the surprising discovery that ahot aqueous solvent extract of tubercle bacillus, by itself, exhibits ananti-HIV effect. Although hot aqueous solvent extract of tuberclebacillus was previously known to promote recovery of white blood cellsby increasing the count thereof after radiotherapy treatments, such ause would clearly not have suggested the surprising new use afforded bythe present invention.

In more detail, the composition which is used in the present invention;i.e., a composition containing as its primary component a polysaccharidederived from a hot aqueous solvent extract of tubercle bacillus, haspreviously shown to recover the white blood cell count that was reducedby radiotherapy for cancers (see, among others, "The Clinical Report(Basic and Clinical Report)," 24(4), 1973(199) and "Nippon IgakuHoshasen Gakkai Zasshi," 50(8) , 993 (1990)).

However, the effect of the present composition, i.e., hot aqueoussolvent extract of tubercle bacillus, on HIV was unknown prior to thepresent invention.

The present composition herein designated "(a)" contains apolysaccharide as its primary component. Preferably, composition (a)contains a polysaccharide whose primary constituents are arabinose,mannose and glucose, as well as a small amount of nucleic acid.Preferably, the polysaccharide has a molecular weight ranging from about5×10² to 5×10⁴ as measured by the gel filtration method. The nucleicacid content of composition (a) is preferably about 0.05-0.3 wt. %. Thecomposition (a) may contain about 1-5 wt. % the protein. Preferably, themannose content of the polysaccharide is about 10-72 wt. %, thearabinose content about 3-30 wt. %, and the glucose content about 5-30wt. %. The composition (a) of particular preference has the mannosecontent about 40-50 wt. %, the arabinose content about 15-25 wt. %, andthe glucose content about 5-15 wt. %.

Composition (a) is obtained by purifying a hot-aqueous solvent extractof tubercle bacillus. More specifically, composition (a) is obtained bysubjecting cells of tubercle bacillus to extraction with hot aqueoussolvent. The extract is then subjected to protein removal treatment andto treatment for removing polysaccharides of molecular weight of about10⁵ or more. The tubercle bacillus may be selected from both types ofhuman Mycobacteria and non-human Mycobacteria, but human Mycobacteriaare preferred. Examples of non-human Mycobacteria include Mycobacteriumbovis, Mycobacterium avium, Mycobacterium microti, Mycobacteriumkansasii, Mycobacterium marium and Mycobacterium intracellulae. Examplesof human Mycobacteria include Aoyama B, H37Rv and H37Ra with Aoyama Bbeing particularly preferred. The extraction with hot aqueous solvent ismore accurately performed with aqueous solvent of a temperature of about80° C. to 120° C. Examples of aqueous solvent include fresh water,saline, sea water and sodium hydroxide solution with fresh water beingpreferred. The protein removal treatment is more thoroughly performed bycausing proteins to precipitate by use of a protein precipitant such assulfosalicyclic acid, trichloroacetic acid, or phosphotungstic acid,followed by the subsequent collection of the supernatant. The treatmentto remove polysaccharides at a molecular weight of 10⁵ or more, ispreferably performed by causing polysaccharides or high molecular weightto precipitate by use of ethanol, methanol, or acetone in a suitableamount, followed by the subsequent collection of the supernatant.

In accordance with the present invention, composition (a) may bedirectly applied to HIV-infected cells so as to act on the cells.Composition (a) is preferably present in an amount of approximately1-1000 μg/ml, with a more particular presence of 5-500 μg/ml, in the invitro system. Also composition (a) is preferably administered byinjection with a preference of a subcutaneous injection, in the in vivocase.

As calculated in terms of the saccharide content of arabinose in the invivo case, the dose of composition (a) is preferably about 2-200 pg perday, more preferably about 20-100 μg per day.

When composition (a) is administered to a subject in need thereof, it ispreferably prepared in the form of pharmaceutical compositions suitablefor the aforementioned administration routes by incorporating thereto agenerally employed, pharmaceutically acceptable carrier. Examples ofcarriers useful for the preparation of pharmaceutical compositionsinclude; vehicles, binders, lubricants, disintegrants, coating agents,emulsifiers, suspensions, solvents, stabilizers, absorption aids, waterfor injection use, and tonicity agents.

Reference to certain examples, which are provided solely for purposes ofillustration and which are not intended to be limitative, will now beused to further describe the present invention.

REFERENTIAL EXAMPLE 1 (Preparation of Composition (a))

Mycobacterium tuberculosis strain Aoyama B, which had been lyophilizedand stored at -20° C., was subjected to seed culture at 37±1° C. in aSauton-potato medium.sup.(1). The resultant cells were inoculated to aproduction medium.sup.(2) and incubated for 5-7 weeks at 37±1° C. Next,the harvested cells were washed with fresh water for injection use,followed by fresh water for injection use being added to the wet cells,at an amount 20 times the weight of the wet cells. To thereby obtain anextract, the mixture was heated to 100° C. for 120 minutes. The extractwas filtered by use of a 0.45 μm-membrane filter and concentrated underreduced pressure so that the saccharide content (converted toD-arabinose by the phenol-sulfuric acid method) fell within the range of4.0-6.0 mg/ml, to thereby obtain a concentrate. Subsequently, in orderto remove proteins, 1%(w/v) sulfosalicylic acid was added to theconcentrate. The mixture was allowed to stand for 15-20 minutes at atemperature no higher than 10° C. Precipitates were removed bycentrifugal separation (at a temperature of 10° C. or lower, 1,150×g, 10minutes), to thereby recover the supernatant. The protein concentrationof the supernatant was not more than 0.30 mg/ml (Lowry method,calculated as tyrosine). The supernatant was further processed to removesulfosalicylic acid until the content of sulfosalicylic acid fell belowthe detection threshold (10 ppm or less, calorimetric method usingferric chloride solution). To have the saccharide content fall withinthe range of 1.8-2.2 mg/ml, the resultant solution was concentratedunder reduced pressure. The concentrate was then combined with sodiumchloride (0.9% (w/v)) and cold ethanol having a volume equal to that ofthe concentrate. The mixture was allowed to stand for no less than 40hours at a temperature no higher than 10° C., then the precipitatedpolysaccharides of high molecular weight were removed by centrifugalseparation (at a temperature of 10° C. or lower, 2,040×g, 10 minutes).Subsequently, the supernatant was combined with four times the volume ofcold ethanol, and the mixture was allowed to stand for no less than 40hours at a temperature no higher than 10° C., to thereby recoverprecipitates. The precipitates were dissolved in water for injectionuse, and after the saccharide content was adjusted to 1.8-2.2 mg/ml, thesolution was subjected to filtration by use of a 0.45 μm membrane filterand to sterilization in an autoclave (121° C., 20 minutes), to therebyprepare a composition (a) solution.

(1) Sauton-potato medium

Washed potato slices were soaked in a Sauton medium, followed bysterilization for 15 minutes at 115° C. The resultant material was usedas a Sauton-potato medium.

    ______________________________________                                        Sauton Medium                                                                 ______________________________________                                        L-asparagine (monohydrate)                                                                              4.0    g                                            Citric acid (monohydrate)        g              2.0                           Magnesium sulfate (heptahydrate)                                                                               g       0.5                                  Potassium monohydrogenphosphate (anhydrate)                                                                 0.5                                                                              g                                            Ammonium iron citrate            g                  0.05                      Glycerol                         ml                                           ______________________________________                                                                         60                                       

The ingredients above were dissolved in water to make a total volume of1,000 ml. By use of sodium hydroxide solution, the pH was adjusted to7.0-7.3.

    ______________________________________                                        (2) Production medium                                                         ______________________________________                                        L-asparagine (monohydrate)                                                                              4.0    g                                            Citric acid (monohydrate)        g             2.0                            Magnesium sulfate (heptahydrate)                                                                               g      0.5                                   Potassium monohydrogenphosphate (anhydrate)                                                                0.5 g                                            Ammonium iron citrate            g                 0.05                       Glycerol                         ml                                           ______________________________________                                                                         60                                       

The ingredients above were dissolved in water to make a total volume of1,000 ml, followed by sterilization in an autoclave (121° C., 20minutes). By use of sodium hydroxide solution, the pH was adjusted to7.0-7.3.

The physicochemical properties of the composition (a) solution were asfollows.

(1) Appearance: Pale yellow clear liquid

(2) pH: 4.50-5.30

(3) Protein content: 3.5 wt. % (as an amino acid, in a freeze-driedproduct)

(4) Nucleic acid content: 0.1 wt. % (in a freeze-dried product)

(5) Constituent monosaccharides of polysaccharide:

Mannose 43.4 wt. %,

Arabinose 18.2 wt. %, and

Glucose 10.4 wt. %.

Methods for determining the constituent monosaccharides ofpolysaccharide:

The polysaccharide was hydrolyzed with 2N trifluoroacetic acid for 2hours at 100° C., and then subjected to liquid chromatography making useof 2-cyanoacetamide fluoroscein derivative (S. Honda, et al., Anal.Chem., 52, 1079 (1980)).

TEST EXAMPLE 1

HIV-1 IIIB (0.01 M.O.I.) was subjected to a two-hour absorption byperipheral blood mononuclear cells (PBMCs, 2×10⁶ cells/ml, derived froma seronegative donor) activated with phytohemagglutinin (PHA), tothereby prepare HIV-infected PBMCs. The HIV-infected PBMCs were culturedin the RPMI-1640 medium supplemented with 20% FCS and 20 U/ml of IL-2 at37° C. in the presence of composition (a). Seven days after theinfection, HIV-1 p24 antigens present in the culture fluids werequantified by ELISA.

As is apparent from the results shown in FIG. 1, the addition of a 100μg/ml dose of composition (a) reduces the amount of HIV-1 p24 antigens,proving that the composition provides an anti-HIV effect.

As may readily be seen, the present invention provides a surprisingability to inhibit replication of HIV.

Having described the present invention, it will now be apparent to oneof ordinary skill in the art that many changes and modifications may bemade to the above-described embodiments without departing from thespirit and the scope of the present invention.

What is claimed is:
 1. A method of inhibiting the replication of HIV,comprising administering to a patient in need thereof an effectiveamount of composition comprising a polysaccharide produced by a hotaqueous solvent extraction of tubercle bacillus,wherein thepolysaccharide is comprised of arabinose, mannose and glucose residues.2. The method of claim 1, wherein the composition further comprisesnucleic acid produced by the hot aqueous solvent extract of tuberclebacillus.
 3. The method of claim 1, wherein the polysaccharide has amolecular weight of about 5×10² -5×10⁴, as determined by gel filtration.4. The method of claim 1, wherein the tubercle bacillus is a humantubercle bacillus.
 5. The method of claim 1, wherein the human tuberclebacillus is an Aoyama B strain.
 6. The method of claim 1, wherein thehot aqueous solvent extraction is conducted at a temperature of about80° C. to 120° C.
 7. The method of claim 1, wherein the compositionfurther about 1 to 5 wt. % of protein.
 8. The method of claim 2, whereinthe composition further contains about 1 to 5 wt. % of protein.
 9. Themethod of claim 1, wherein the polysaccharide is comprised of 10-72 wt.% mannose, 3-30 wt. % of arabinose and 5-30% wt. % of glucose.
 10. Themethod of claim 1, wherein the polysaccharide is comprised of 10-72 wt.% mannose, 3-30 wt. % of arabinose and 5-30% wt. % of glucose.
 11. Themethod of claim 1, wherein the aqueous solvent is fresh water, saline,sea water, or sodium hydroxide solution.
 12. The method of claim 1,wherein the aqueous solvent is fresh water.
 13. The method of claim 1,wherein the composition is obtained by a process comprising:(a)extracting tubercle bacillus cells with an aqueous solvent at atemperature of at least about 80° C., to obtain a concentrate whichcomprises protein and polysaccharide; (b) precipitating protein in theconcentrate from (a), to produce a precipitate and a supernatantcomprising polysaccharide; and (c) precipitating polysaccharide in thesupernatant from (b); and (d) isolating the precipitated polysaccharidefrom (c), followed by dissolving the precipitated polysaccharide inwater.